In clinical applications, in research and in industry, a need exists to detect the presence of minute amounts of organic material, such as antigens, antibodies, hormones, metabolites, enzymes, and drugs. In clinical situations, detecting the presence or absence of metabolites, hormones, or other organic factors in serum or in other body fluids may be useful in the diagnosis of many clinical conditions, such as pregnancy, infection, blood disorders, hepatitis, etc. The detection and quantitative analysis of organic agents is often required in immunological, biological, chemical, or other types of scientific and medical research. In industry, assays for organic materials are utilized in quality control procedures for the production of chemicals and in monitoring the pollution of water.
Of the numerous chemical and biological assays that have been developed to detect organic materials, of relevance to the present invention are precipitation and agglutination assays. In a typical precipitation assay, the organic material interacts with a reactant to form a complex that falls out of solution. In agglutination reactions, the organic substance of interest cross-links an insoluble reactant to cause the reactant to flocculate. Optical scattering techniques are commonly used to measure the flocculation. A drawback of precipitation and agglutination assays is that they are not as sensitive as radioimmunoassays. Also, although optical techniques have been developed to improve the sensitivity of these assays, these techniques require specialized equipment and analysis.
Another type of known assay for organic materials involves labeling either the organic material or a reactant thereto with a radioactive, fluorescent or other type of tracer substance to ascertain the extent to which the organic material has coupled with its reactant.
Radioimmunoassay is one of the most common types of these "tracer" assays. Radioimmunoassay involves combining a known amount of radiolabeled organic material with a sample containing an unknown amount of unlabeled organic material of interest together with a specific antibody that binds indiscriminantly to the labeled and unlabeled organic materials to form a complex. After an incubation period, the unbound organic materials are separated from the bound organic materials, typically by precipitation of the complex with polyethylene glycol, adsorption of the unbound material with activated charcoal or utilization of solid-phase reagents. Then the radioactivity of either of these two fractions is measured. A certain amount of the labeled and unlabeled organic material will be bound to the reactant, with the amount of the bound labeled organic material being inversely related to the quantity of unlabeled inorganic material present in the sample being tested.
A drawback of the radioimmunoassay is that the procedures for separating the bound organic materials from the unbound require a significant number of time-consuming operations that are often complicated and expensive. The separation procedures involve repeatedly washing the complex of organic material and antibody with a rinsing solution and/or centrifuging the mixture to remove the unbound organic material from the reactant, thereby generating radioactive waste material with each washing. By the time that the separation process has been completed, significant volumes of radioactive waste material are produced. This waste material is not only expensive to dispose of, but also presents a potential health hazard to persons handling the material, including during the separation procedures.
In an assay utilizing fluorescence, the organic material may be labeled with an appropriate fluorescer, such as fluorescein isothiocyanate. The extent to which the fluorescer labeled organic materials bound to a specific reactant can be examined under a light microscope with a suitable light source and filters to provide incident light of the proper wavelength to cause fluorescence.
An example of a particular fluorescence technique is disclosed in U.S. Pat. No. 4,161,515 wherein an unknown organic compound, whose presence is being investigated, is mixed with: (1) a known quantity of antibody against the organic compound; (2) an organic analog, having a fluorescer bound thereto, which competes with the unknown organic compound for the antibody; and (3) an antibody for the fluorescer. The competition between the unknown organic material and the known analog-fluorescer effectively reduces the concentration of the antibody, thereby causing more of the fluorescer-antibody to combine with the analog-fluorescer. This, in turn, causes a corresponding change in the emission spectrum of the fluorescer.
A tracer assay that utilizes both fluorescence and radioactive substances is disclosed by U.S. Pat. No. 4,000,252 wherein a known quantity of radiolabeled antigen and a sample containing an unknown amount of unlabeled antigen are placed within an immunoscintillation cell. An insolubilized or solid phosphor, which is chemically or physically associated with an antibody to the antigens, is also added to the cell. The unbound antigens are washed from the cell and then the luminescence emitted by the phosphor due to activation from the radioactive energy from the found labeled antigens is measured inside a scintillation counter. Removal of the unbound antibodies from the cell requires several washing procedures that are not only time consuming, but also produce significant quantities of radioactive waste material.
Assays that combine tracer techniques with agglutination are disclosed by U.S. Pat. Nos. 4,018,972 and 4,271,139. In U.S. Pat. No. 4,108,972, microscopic carrier particles, each containing a fluorescent tracer material, and a biological reactant to the antibody or antigen being investigated, are placed in suspension. When the antigen or antibody is added to the suspension, it binds to the reactant coating to cause flocculation of the carrier particles. The flocculated material is then separated from the suspension fluid and other constituents by numerous washings. Thereafter, the flocculated material is dissolved and then assayed by flourescence techniques to determine the quantity of organic material present.
In U.S. Pat. No. 4,271,139, tritiated (radioactive) latex particles coated with antigen and polystyrene scintillant particles coated with the same antigen were placed in an aqueous medium with a sample containing an unknown quantity of a corresponding antibody. The number of tritiated antigen coated latex particles linked to the antigen coated scintillant particles is related to the concentration of antibody present. Also, when the two particles are linked together by the antibody, the radioactive energy from the tritiated particles initiates scintillation within the scintillant particles. Scintillations are then measured by an appropriate detector, with the detected level of scintillation being indicative of the quantity of antibody present. Addition of an unknown quantity of non-radioactive antigen then competes with the antigen coated bead binding to the antibody, thereby reducing bead agglutination, scintillation and signal. A drawback of this particular assay is that it requires both tritiated latex particles and polystyrene scintillant particles to be coated with antigen, which increases the expense and complexity of the assay. In addition, relative to the standard radioimmunoassay discussed above, extremely large suspension volumes are required for the assay to operate properly. The assay process of the '139 patent also requires the availability of relatively pure antigen to be used to bind to the two types of carrier particles. Relatively pure samples of antigen are both expensive and difficult to obtain.
Thus, it is a principal object of the present invention to provide an accurate, inexpensive, and rapid assay procedure and test kit for detecting the presence of extremely small amounts of organic materials.
A particular object of the present invention is to provide an assay of equivalent accuracy to present techniques, but which does not require highly skilled personnel or large amounts of time to perform using standard commercially available equipment.
A further particular, but highly important object of the present invention, is to provide an assay procedure that produces only a minimum volume of radioactive waste and requires only a minimum amount of handling of hazardous substances.
An additional particular object of the present invention is to provide an assay that can be used to rapidly test a large number of samples.
Another particular object of the present invention is to provide an assay that utilizes water as a suspension medium.